Trace elements such as selenium (Se) are considered to be essential for life. This element provides significant health benefits for both animals and humans; however, it must be continuously ingested through the diet as mammals have a limited capacity to accumulate this element. Selenium can be found naturally in the ground and can enter the food chain by consumption of water and plants. The amount of selenium found in plants depends on the geography, climate and the geology of the ground where the plants were grown or cultured.
Selenium can be supplemented in water and food, in different chemical forms such as in organic or inorganic matter. The most common organic form of selenium is found in seleno-aminoacids such as seleno-methionine (NH2CH(CH2CH2SeCH3)CO2H), seleno-cysteine (NH2CH(CH2SeH)—CO2H), and seleno-methyl-selenocysteine (SMSeC) (NH2CH(CH2SeCH3)CO2H), while the most common inorganic form is found in various selenite (SeO3−2) and selenate (SeO4−2) inorganic salts, for example, sodium selenite (Na2SeO3) and sodium selenate (Na2SeO4). Seleno-methyl-selenocysteine is a naturally occurring seleno-amino acid that is synthesized by plants such as garlic, astragalus, onions and broccoli. Unlike seleno-methionine, which is incorporated into proteins in place of methionine, seleno-methyl-selenocysteine is not incorporated into any proteins, thereby being fully available for the synthesis of selenium-containing enzymes such as glutathione peroxidase.
The effect of selenium on human health depends on the dose and chemical form being ingested. The concentration range within which this element is considered toxic or beneficial is very narrow. It is estimated that a food diet that contains more than 1 mg of selenium per kg could produce toxic effects, while a food diet with a concentration of less than 0.1 mg per kg of selenium may produce a state of deficit of this element. The recommended daily intake of selenium is an average of 55 μg for adult women and 70 μg for adult men, while the Maximum Tolerable Intake is 400 μg/day for these age groups. Selenium also has an effect when administered in chemopreventive supra-nutritional doses, defined as doses higher than the requirements recognized as being adequate from a nutritional standpoint, i.e. the recommended daily intake. This effect depends strongly on the chemical form of the trace element. Compared with other organic and inorganic forms, the organic form of seleno-methyl selenocysteine offers the greatest effect of chemoprevention.
The toxicity of selenium depends on its chemical form, oxidation state and dose and is manifested as either acute or chronic selenosis. Acute exposure to selenium can cause bronchitis, pneumonia and pulmonary edema, whereas chronic exposure produces discoloration of the skin, hair loss, deformed nails, and weakened attention. In general, the damage caused by an excess of selenium is much less severe than that caused by a deficiency of this element. Selenium deficiency causes a reduction in the expression of selenoproteins, and thus alters all metabolic processes mediated by them. There are some endemic sicknesses in some selenium-deficient regions such as China and eastern Siberia, among which are, Keshan disease (cardiomyopathy) and Kashin-Beck disease (a type of arthritis deformans), plus a form of cretinism (congenital hypothyroidism) associated with low selenium metabolic status. Selenium deficiency has also been associated as the cause of immune system problems, male infertility, development of some types of cancer, and cardiovascular diseases. It has been shown that increased selenium intake results in improved immune responses. On the other hand, it has demonstrated there is a strong correlation between the risk of developing cardiovascular disease and low metabolic status of selenium in humans.
The metabolic state of the trace element selenium depends on the amount and chemical form in which this element has been administered. These characteristics depend on the geographic location and soil characteristics existent were the food has been grown or produced such as (meat and vegetables), in many cases it may not be adequate to meet the needs of the organism that consumes them and still be well below those required to achieve the beneficial health effects, such as prevention of cancer, immune system stimulation, increased fertility in men, and improving the antioxidant defence of cells. Against this, it is necessary to have a procedure to determine whether the intake of organic selenium, as well as other trace elements in an individual or group of individuals, is adequate to ensure the health benefits mentioned above.
Currently, there are no specific procedures for determining the metabolic state of organic forms of selenium in mammals. Traditional procedures for measuring selenium in tissues or body fluids, such as the total concentration of selenium in blood plasma, the enzyme activity of glutathione peroxidase on erythrocytes and the concentration of plasma selenoprotein P, which are not capable to distinguish between organic forms (which are cancer preventive) and inorganic forms (which can even be toxic), nor between the recommended selenium levels and supra-nutritional levels (which provide the above mentioned health benefits). Thus, it is not possible to determine whether an individual has received the appropriate dose of organic selenium, and consequently it is not possible to have an idea of the level the individual needs for their protection against certain diseases including some types of cancer.
There are analytical methods that are currently being used to determine the metabolic status of selenium in mammals, which are accepted by the scientific community and have been used in several epidemiological studies on selenium. These methods are described below.
To determine the total concentration of selenium in blood plasma, plasma can be obtained from a heparinized blood sample, taking care not to use EDTA or other chelating agents, and the concentration of selenium can be measured by atomic absorption in a spectroscopy graphite furnace.
The activity of the enzyme glutathione peroxidise in whole blood, blood plasma, and also in erythrocytes can be measured by carrying out the reaction at 25° C. in the presence of glutathione reductase and NADPH, using tert-butyl hydroperoxide as the substrate, and the change in absorbance at 340 nm can be recorded for 4 to 5 minutes.
The concentration of selenoprotein P in plasma can be measured using different methods. These methods include immunodetection or “western blot” and ELISA (enzyme-linked immunosorbent assay) procedures. The effect of selenium on health can be quantified by measuring the level of expression of selenoprotein P, typically by measuring the concentration of mRNA by real-time PCR.
The three procedures described above are based on the determination of a species or a particular protein, and each of them alone does not provide sufficient information to establish the metabolic status of selenium, but rather uses a combination of them. It has also been reported that these methods give the same results for different chemical forms of selenium contained in the diet. However, these methods are not able to distinguish between adequate selenium nutritional status and the status in the supranutritional state.
It should be kept in mind that there were no patents or patent applications in Chile, related to analytical methodologies procedures or diagnostic procedures for the determination of the presence of selenium in the National Institute of Industrial Property of Chile (INAPI). On the other hand, worldwide, there are patent applications and granted patents relating to the determination of selenium and the identification of specific proteins for diagnosis purposes. These patents are described below.
There are several procedures and kits for determining the concentration of selenium, which are based on the use of proteins or enzymes and are protected by patents in USA, Europe, and/or Japan. For example, U.S. Pat. No. 6,884,601 relates to a method for determining the sperm fertilizing potential, based on measuring the activity of phospholipid-hydroperoxide selenoenzyme-glutathione peroxidase. European Application RU 19940027650 relates to a biosensor which corresponds to biochemical reagents in which a type reductase enzyme that induces a chemical reaction when in the presence of metal ions such as selenium. U.S. Pat. No. 5,830,673 relates to a bioassay to detect the presence of selenium and quantify its abundance, by using the microorganism Escherichia coli, in which a plasmid encoding beta-galactosidase dependent on selenium has introduced. It has been applied in biological samples, either blood or food, where bacterial growth was observed only if there is availability of selenium in the sample. Finally, U.S. Pat. Nos. 6,849,417 and 7,422,543 relate to a 15-kDa selenoprotein, whose gene polymorphisms are associated with the development of prostate cancer. This information can be used as an indicator of the need for dietary supplementation containing selenium to inhibit tumor development in susceptible individuals. While the latter references are not focused on the determination of selenium, they do relate to this request, as implicitly indicated, the need to quantify the nutritional status of selenium in an individual.
There are several procedures and kits for determining the concentration of selenium, which are based on chemical techniques. Japanese Patent Application JP19970302381 refers to a method for separating and determining hexavalent selenium from quadrivalent selenium in aqueous solution by adding hydrochloric acid (35%) and nitric acid (61%). European Patent Application RO19920000879 relates to a spectrophotometric method for the determination of selenium in spirulin biomass, which includes acid digestion of biomass and the removal of all ions with the exception of selenium ions from the sample. European Patent Application No. CS19880002378 refers to a method for determining trace concentrations of selenium in biological material via neutron activation analysis (“Activating Neutron Analysis”); and European Patent Application No. RU19930045944 refers to a method for the determination of selenium in organic and inorganic substances, for which application there is no abstract available.
Additionally, there are applications and patents which are related to the proposed alternative procedures of this application, which refer to the detection of diseases and counselling preventive nutrition. U.S. Patent Application No. 2004/0126822 is directed to a method and a kit for diagnosing diseases involving an inflammatory reaction, and is based on the determination of the concentration of selenoprotein P in blood plasma of a patient, and in the comparison with a reference or patron sample, where the detection and quantification of the protein was performed using antibodies. U.S. Pat. No. 7,288,274 refers to a process for detecting oxidative stress and kits for its implementation, that applies to individuals who pose a risk factor (such as certain diseases or habits), and it consists in the selection and quantification of at least two markers of oxidative stress, and compare their level on the individual at risk level in comparison with the level in reference individuals. These markers can be proteins and enzymes, related to oxidative stress, and to trace elements such as selenium, among other. U.S. Patent Application No. 2009/0081685, is directed to methods and compositions for detecting ovarian cancer in early stages of development of the disease, based on the overexpression of at least one characteristic of the disease biomarker (such as haptoglobin, gelsolin, transthyretin, carbon anhydrase, among other) in the blood serum of the patient. U.S. Patent Application No. 2005/0240436, relates to a method for creating an individual recommendation for nutritional supplementation focused on micronutrients, based on at least one of the three groups of body indicators and by comparing the value obtained with the expected values in normal individuals, this determines whether the supplementation is necessary or not. For this purpose a database that generates the corresponding recommendations, is used. Of the documents found in the search carried out, none of them is related to the procedure proposed and developed in the present invention of this patent application.